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Abstract

Background

Carbohydrate restricted diets (CRD) consistently lower glucose and insulin levels
and improve atherogenic dyslipidemia [decreasing triglycerides and increasing HDL
cholesterol (HDL-C)]. We have previously shown that male subjects following a CRD
experienced significant increases in HDL-C only if they were consuming a higher intake
of cholesterol provided by eggs compared to those individuals who were taking lower
concentrations of dietary cholesterol. Here, as a follow up of our previous study,
we examined the effects of eggs (a source of both dietary cholesterol and lutein)
on adiponectin, a marker of insulin sensitivity, and on inflammatory markers in the
context of a CRD.

Results

Body weight, percent total body fat and trunk fat were reduced for all subjects after
12 wk (P < 0.0001). Increases in adiponectin were also observed (P < 0.01). Subjects
in the EGG group had a 21% increase in this adipokine compared to a 7% increase in
the SUB group (P < 0.05). Plasma CRP was significantly decreased only in the EGG group
(P < 0.05). MCP-1 levels were decreased for the SUB group (P < 0.001), but unchanged
in the EGG group. VCAM-1, ICAM-1, TNF-α, and IL-8 were not modified by CRD or eggs.

Conclusion

A CRD with daily intake of eggs decreased plasma CRP and increased plasma adiponectin
compared to a CRD without eggs. These findings indicate that eggs make a significant
contribution to the anti-inflammatory effects of CRD, possibly due to the presence
of cholesterol, which increases HDL-C and to the antioxidant lutein which modulates
certain inflammatory responses.

Background

Insulin resistance is recognized as the major defect leading to the development of
the metabolic syndrome including a proinflammatory state. Experimental and epidemiological
evidence reveals that chronic inflammation is an independent predictor of cardiovascular
disease (CVD) [1] and is directly involved in the promotion of insulin resistance and atherosclerosis
[2-4]. Infections or the healing of wounds produce an inflammatory response which attracts
leukocytes to a localized area of the vasculature and permits passage through the
underlying tissue [5]. Repeated stimulation of this inflammatory response can lead to the development of
several chronic diseases mediated by increased levels of cytokines and chemokines.
Adipose tissue secretes several adipokines, such as tumor necrosis factor-alpha (TNF-α),
interleukin-8 (IL-8), and adiponectin which can modulate lipid and glucose metabolism
[6]. Adiponectin is a unique hormone that is both anti-inflammatory and anti-atherogenic
and low circulating levels are found in obese, insulin resistant individuals. Elevated
levels of the acute-phase reactant C-reactive protein (CRP) and TNF-α are associated
with an increased risk for numerous chronic diseases [7]. Monocyte chemoattractant protein-1 (MCP-1) is a proinflammatory chemokine produced
in response to inflammatory stimuli like TNF-α. Additionally, upregulation of vascular
cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) signifies
the initiation of atherogenesis by permitting circulating cells to adhere to the endothelium
[8-10].

Acute carbohydrate consumption has been shown to stimulate reactive oxidative species
while initiating many pro-inflammatory processes [11]. Additionally, isocaloric high carbohydrate diets are associated with several markers
of inflammation [12]. Carbohydrate restricted diets (CRD) are able to reduce biomarkers of inflammation
in the absence of weight loss [13,14]. Dietary carbohydrate, rather than fat, plays a critical role in activating pro-inflammatory
processes through their effect on the fatty acid composition of lipids and membranes
[15]. Endogenous fatty acids function as ligands for receptors and transcription factors
that modulate inflammatory signaling cascades. CRDs are ideal for reducing inflammation
because they reduce plasma glucose excursions, the major stimulus for pancreatic secretion
of insulin, and modulate the underlying factors associated with CVD and the metabolic
syndrome.

Eggs are a breakfast staple that are affordable and provide a good source of protein
and other valuable nutrients. Experimental studies show that the additional dietary
cholesterol provided by whole eggs does not increase the risk of CVD in a variety
of sample populations [16,17]. Additionally, whole eggs contain the potent antioxidant, lutein, which protects
against numerous inflammatory processes [18]. To our knowledge this is the first study measuring inflammatory marker responses
to a CRD utilizing eggs as a source of dietary cholesterol and lutein.

We have previously shown that during an intervention following a CRD, adult men consuming
eggs presented a significant increase in plasma HDL-C (P < 0.0001) compared to those
individuals who were consuming egg substitute [19]. The purpose of this study was to determine whether the observed increases in HDL-C
[19], plus the fact that eggs are a good source of bioavailable lutein [17], would affect the inflammatory response. Our initial hypothesis was that the additional
dietary cholesterol provided by eggs would not modify the beneficial effects of the
CRD.

Subjects

Thirty-one healthy men aged 40–70 y, with a BMI between 25 and 37 kg/m2 were recruited from the university and the surrounding community[19]. All subjects gave a written informed consent to participate and the Committee on
the Use of Human Subjects in Research of the University of Connecticut approved the
study protocol.

Study Design

The CRD was similar to those implemented in our previous studies[14], we conducted this study to determine the effects of egg intake in conjunction with
a CRD on the variables for the classification of the metabolic syndrome [19] and on body composition and inflammation markers presented in this manuscript. Briefly,
subjects (n = 28) were matched by age and BMI (body mass index) and then randomly
assigned, single blinded, to eggs (EGG, n = 15) (640 mg cholesterol) or placebo (SUB,
n = 13) (no added cholesterol) for twelve weeks. Both products were equal in consistency
and color. All subjects attended a group meeting before the intervention with registered
dieticians where detailed instructions on keeping diet records and following a CRD
similar to those used in our previous studies [14,20] were discussed. This was a free living food study and no food was provided to subjects.
The diet was designed to restrict carbohydrates (~10% of total calories) so that ketones
are produced. Subjects were also instructed to abstain from consuming eggs (other
than those provided by the study) during the whole intervention. To monitor compliance
to the ketogenic diet, subjects used Ketostix reagent strips to assess ketonuria nightly
and returned their empty egg containers weekly. Subjects were asked to maintain their
habitual level of exercise throughout the intervention. One week prior to the start
of the study, subjects completed a 3 day food record to assess habitual food intake.
Blood samples, DEXA, food records and anthropometrics were collected at baseline and
week 12.

Blood collection

After an overnight fast, blood was collected into EDTA from an antecubital vein. Plasma
was separated by centrifugation at 2000 × g; 20 min, and aprotinin (0.5 mL/100 mL),
sodium azide (0.1 mL/100 mL), and phenylmethylsulfonyl fluoride (0.1 mL/100 mL) were
added for preservation. Plasma was stored in individual aliquots at -80°C for analysis
of cytokines.

Plasma lipids, lutein, and insulin

Plasma triglycerides and total cholesterol were measured by enzymatic analysis and
HDL-cholesterol by measuring cholesterol in the supernatant following precipitation
of the apo B containing lipoproteins as previously reported [16]. LDL-C was estimated by the Friedwald equation [21]. Plasma lutein was measured using Waters' HPLC system [22] and detection was measured at 450 nm. Insulin was measured with multiplex assay kit
based on the Luminex × MAP technology (Linco Research, Inc, St. Charles, MI).

Body Weight and DEXA

Weight was measured to the closest 0.25 kg and height to the closest 1 cm on a portable
stadiometer/scale. Body mass and body composition were measured in the morning after
an overnight fast. Body mass was recorded to the nearest 100 g on a calibrated digital
scale with subjects wearing only underwear. Whole body and regional body composition
was assessed using a state-of-the-art fan-beam dual-energy X-ray absorptiometry (Prodigy™,
Lunar Corporation, Madison, WI). Analyses were performed by the same blinded technician.

Fasting Adiponectin, sVCAM-1 and sICAM-1

From fasting plasma, VCAM-1, ICAM-1, and adiponectin were measured in duplicate in
the same assay using the Human CVD Panel 1 Lincoplex kit. Samples were diluted 1:100
and simultaneously quantified by using Antibody-Immobilized beads and Luminex × MAP
technology. All assays were carried out in the same day to decrease variability. The
CV was between 2–6%. The sensitivity for VCAM-1, ICAM-1, and adiponectin were 16.0
pg/ml, 9.0 pg/ml, and 56.0 pg/ml, respectively.

Fasting TNF-α, IL-8, and MCP-1

Plasma TNF-α, IL-8, and MCP-1 concentrations were measured in duplicate in the same
assay from a fasting sample using the Human Cytokine Lincoplex kit, which is a multiplex
assay kit based on the Luminex × MAP technology (Linco Research, Inc, St. Charles,
MI)[23]. This method uses Antibody-Immobilized beads for simultaneous quantification of TNF-α,
IL-8, and MCP-1. All assays were carried out in the same day to decrease variability.
The CV was between 3–6%. The sensitivity for this assay was 0.66 pg/ml, 1.12 pg/ml,
and 1.29 pg/ml, respectively.

Fasting CRP

CRP concentration was measured in duplicate from fasting plasma with a 1:2000 dilution
using the Human CVD Panel 2 (acute-phase proteins) Lincoplex kit. Antibody-Immobilized
beads were analyzed using Luminex × MAP technology. The sensitivity of this assay
was 6.0 pg/ml.

Statistical Analysis

Data are presented as means ± SD. A two-way repeated measures ANOVA was used to determine
the effects on each subject on all parameters. Each individual's response to the intervention
over time was considered as the repeated measure and egg vs. egg substitute the between
subject factors. Pearson correlations were used when appropriate and differences of
P < 0.05 were considered significant.

Results

Diet and Plasma Lipids

In spite of the ad libitum dietary intervention, caloric intake decreased significantly
for all subjects (n = 28) from 10243 ± 4039 kJ at baseline to 7967.82 ± 2401 kJ at
week 12 (P < 0.05). There were significant changes in macronutrient intake during
the intervention. Carbohydrate intake decreased from 42% of total calories at baseline
to 17% at week 12 (P < 0.001). Protein intake increased from 17.8% at baseline to
25.8% at week 12 (P < 0.001). Fat intake increased from 39.6% of total energy at baseline
to 55.6% at week 12 (P < 0.001). These changes were consistent among the 28 subjects
irrespective of the dietary group. However, dietary cholesterol increased from 319
± 150 g/d at baseline to 827 ± 192 g/d at week 12 (P < 0.001) for the EGG group while
there were no changes in dietary cholesterol for the SUB group. Values were 354 ±
170 g/d at baseline and 277 ± 100 g/d after 12 wk (P > 0.15). A more detailed description
of the diet has been previously reported [19]

There was a significant increase in HDL-C for subjects in the EGG group after 12 wk
of the intervention (P < 0.001) from 47.6 ± 15,1 mg/dL to 57.1 ± 15.1 mg/dL while
the subjects in the SUB group did not change their HDL-C (50.0 ± 9.1 mg/dL at baseline
and 48.9 ± 8.8 mg/dL post-intervention). Further, 13 out of 15 subjects had an increase
in HDL-C in the EGG group while only 3 out of 13 subjects had an increase in the SUB
group [19]. Plasma triglycerides were decreased for all subjects by an average of 45% (P < 0.0001).
The combined values for triglycerides were 120.2 ± 59.4 mg/dl at baseline and 73.4
± 26.9 mg/dl after week 12. There were no changes in total cholesterol observed in
either group (193.3 ± 37.9 mg/dl at baseline and 194.8 ± 40.7 mg/dl at week 12). Individual
responses for total cholesterol between baseline and 12 weeks are presented in Figure
1 for both the EGG and the SUB groups. There was a great variation in the response
to diet going from + 42 mg/dL to -74 mg/dl changes in total cholesterol between baseline
and 12 wk. There were no significant changes in LDL-C between baseline and post-treatment
(P > 0.05) for either group. Values were 117.8 ± 37.8 mg/dl at baseline and 132.9
± 43.6 mg/dl after week 12.

Figure 1.Changes in total cholesterol between baseline and post-treatment for subjects from
the EGG group (blue lines, n = 15) and the SUB group (red lines, n = 13). The pink line represents the mean for the group.

Plasma and Dietary Lutein

Plasma lutein was only increased in the EGG group (P < 0.01) from 0.54 ± 0.24 μmol/L
to 0.93 ± 0.42 μmol/L while there were no changes in plasma lutein for the SUB group
(0.53 ± 0.29 μmol/L at baseline to 0.53 ± 0.35 μmol/L after 12 wk)

Anthropometrics and Insulin

Body weight and fat mass were decreased significantly for both the EGG and SUB group
after the intervention (P < 0.0001) (Table 1). Body weight decreased 6.7 kg and 5.9 kg for the EGG and SUB groups, respectively.
Likewise percent body fat and percent trunk fat were significantly decreased for both
groups after 12 wk (P < 0.0001). Changes in plasma insulin between baseline and 12
wk for the EGG and SUB groups are presented in Figure 2, panel A. Individual changes for all subjects are presented in Figure 2, Panel B. Changes in insulin went from an increase of 51 pmol/L to a decrease of -114 pmol/L
with a mean change of -26 pmol/L.

Table 1. Body weight and body composition as measured by DEXA at Baseline and following 6 and
12 weeks of a carbohydrate restricted diet with 3 eggs per day (EGG group) or the
equivalent of egg substitute (SUB)1

Figure 2.Concentrations of insulin for subjects from the EGG (n = 15) or the SUB (n = 13) groups
at baseline and at week 12. ** indicates significantly different from baseline (Panel A). Individual responses
to insulin are indicated in Panel B for subjects from the EGG group (blue lines, n
= 15) and the SUB group (red lines, n = 13). The pink line represents the mean for
the group

Adiponectin, CRP, VCAM-1, and ICAM-1

The fasting values of adiponectin, CRP, VCAM-1, ICAM-1, are presented in Table 2. Adiponectin was significantly increased (P < 0.01) for both groups, with a higher
increase in the EGG (15.3 g/L to 18.5 g/L) (P < 0.05). The individual changes in adiponectin
for all subjects (n = 28) are presented in Figure 3. There was an increase in adiponectin in 12 subjects from the EGG group from a total
of 15 while only 7 out of 13 subjects from the SUB group presented an increase in
adiponectin (Figure 3). CRP was only decreased in the EGG group (P < 0.05) Values were reduced from 5.95
mg/L to 4.33 mg/L while there was a non-significant increase in the SUB group (Table
2). There were no differences in ICAM-1 or VCAM-1 observed in either group.

Table 2. Fasting serum Adiponectin, CRP, VCAM-1, ICAM-1 at Baseline and following 12 weeks
of a carbohydrate restricted diet with 3 eggs per day (EGG group) or egg substitute
(SUB).

Figure 3.Changes in adiponectin between baseline and post treatment for subjects from the EGG
(panel A, n = 15) or the SUB (panel B, n = 13) group. The red line represents the
mean for the group.

TNF-α, IL-8, and MCP-1

The fasting values of TNF-α, IL-8, and MCP-1 before and after the intervention are
presented in Table 3. MCP-1 was significantly reduced (P < 0.05) for the SUB group after the intervention,
while no change was observed in the EGG group (interaction effect, P < 0.05). There
were no changes in TNF-α or IL-8 observed in either group.

Table 3. Fasting serum TNF-α, IL-8 and MCP-1 concentrations at Baseline and following 6 and
12 weeks of a carbohydrate restricted diet with 3 eggs per day (EGG group) or placebo
(SUB).

Discussion

In this study we are reporting for the first time that eggs modulate the response
of certain inflammatory cytokines during a weight loss intervention using a CRD. Subjects
consuming the eggs presented a better response to adiponectin and CRP, two major markers
of inflammation and of CHD risk. This effect could be due to the high concentration
of lutein, a potent antioxidant present in the egg yolk.

Adiponectin

There was a significant increase in adiponectin in both the EGG and SUB groups, with
a greater increase in adiponectin observed in the EGG group. An anti-atherogenic adipokine,
adiponectin decreases adhesion molecule expression that occurs after inflammation
[24] and also decreases TNF-α production by macrophages [25]. Reductions in weight and fat mass in obese individuals is often accompanied by an
increase in serum adiponectin [26], accordingly, we observed a significant reduction in trunk fat in this study following
the intervention. Inflammatory cytokines suppress adiponectin expression [27], so the increase in adiponectin may also be directly related to reduction in these
inflammatory markers and because of a selective decrease of CRP only by eggs, a higher
increase of adiponectin was observed for this group. Paraoxonase is an antioxidant
carried on HDL that protects against LDL oxidation [28], and with inflammation, there is a reduction in paroxonase levels [29]. The increased HDL-C and additional adiponectin observed in the EGG group may have
resulted in elevations of paraoxonase, which may explain improvements in the inflammatory
profile. Direct administration of adiponectin stimulates fatty acid oxidation in hepatic
and muscle tissues, which leads to decreased triglycerides [30]. Increased adiponectin levels are associated with increased insulin sensitivity,
decreased triglycerides, and increased HDL-C [31]. Accordingly, we found that the EGG group presented a significant increase in plasma
HDL-C in addition to higher levels of adiponectin. Egg intake is associated with significant
increases in HDL-C and concentration of anti-atherogenic large HDL particles[16,17]. Large HDL particles are correlated with decreased CHD risk [17,32] because they can more efficiently remove excess cholesterol by returning it to the
liver for excretion. It is postulated that adiponectin may influence reverse cholesterol
transport (RCT) by increasing HDL production in the liver by promoting apoA-1 synthesis
and the ABCA1 pathway [33]. Additionally, accelerated RCT results in decreased secretion of ApoB-100 containing
lipoproteins [34], which is evidenced by the reduction in triglycerides. Lipoprotein lipase (LPL) catalyzes
the hydrolysis of triglycerides from ApoB-100 containing particles. Several studies
show that adiponectin is highly correlated with LPL activity [35,36]. In addition, there were significant reductions in insulin for both groups. Carbohydrate
restriction improves insulin resistance by lowering insulin levels and the disinhibition
of hormone sensitive lipase, promoting triacylglycerol hydrolysis [13]. As a result, there is an increase in cellular fatty acid uptake and oxidation [13]. The increase in fatty acid oxidation decreases hepatic triglyceride production and
the synthesis and secretion of triglyceride-rich VLDL particles. Taken together, these
data suggest that interventions which increase HDL may also be directly involved in
improving the inflammatory response and increasing insulin sensitivity as demonstrated
by the increased levels of plasma adiponectin.

CRP

At week 12, CRP was reduced only in the EGG group. Serum CRP is an important marker
of vascular inflammation and can be used to predict atherosclerosis [7]. Reduced CRP levels are found after weight loss interventions and are inversely correlated
to adiponectin [37]. The reduction in adipose tissue due to weight loss reduces the number of pro-inflammatory
cytokines (TNF-α, IL-8.) secreted by monocytes, and the number of macrophages and
endothelial cells present in the adipocytes [7]. Macrophages and endothelial cells produce CRP, so the reductions in adipose tissue
as well as a reduction of carbohydrates work in parallel to mediate the reduction
in CRP. Previously, Tannock et al. [38], found that the addition of 4 whole eggs/day for 4 weeks was associated with increased
levels of CRP in lean, insulin sensitive individuals, however, there was no effect
on CRP in obese or insulin resistant individuals. The subjects in the current study
were obese individuals undergoing weight loss and we found that additional dietary
cholesterol lowered CRP levels. In previous studies, diets high in monounsaturated
fatty acids and polyunsaturated fatty acids have been associated with decreased serum
CRP [39]. Additionally, eggs contain the potent antioxidants lutein and xeazanthin, which
might play a role in the reduction of inflammation observed in the EGG group. Both
groups had significant increases in dietary intake of lutein, but only the EGG group
experienced an increase in plasma. These findings correspond with results from another
study that found increases in the size and number of HDL particles is associated with
increased plasma lutein [18]. We speculate that the reduction of CRP in the EGG group may be the result of increased
HDL-C preventing CRP induced upregulation of inflammatory adhesion molecules, possibly
through enhanced paraoxonase activity from antioxidants and fatty acids contained
within the egg yolk, which can break down oxidized lipids to counteract proinflammatory
effects.

TNF-α, IL-8, and MCP-1

TNF-α is an inflammatory cytokine that is secreted 7.5 times higher from the abdominal
adipose tissue of obese subjects than their lean counterparts [40]. A few experimental studies show that weight loss through short-term caloric restriction
can reduce circulating levels of TNF-α [14,41], however a reduction of fat mass does not always signal a reduction in TNF-α [42,43]. We did not observe a change in fasting TNF-α in either experimental group. These
discrepancies are likely the result of the duration and method of weight loss. Further
studies are needed to understand the relationship between TNF-α and central obesity.
Additionally, IL-8 did not change after the intervention either. Stimulated by oxidized
low-density lipoprotein, IL-8's released from macrophages plays a key role in the
development of atherosclerosis [44]. IL-8 allows adhesion of monocytes to the endothelium [45], which promotes the atherosclerotic process. TNF-α stimulates IL-8 synthesis in the
adipocytes [46], so the equivocal results found for IL-8 production likely relates to the failure
of TNF-α levels to change after the intervention.

MCP-1 levels were significantly reduced for the SUB group at week 12, while no change
was observed for the EGG group. MCP-1 is an inflammatory chemokine mostly produced
by endothelial cells and macrophages. In visceral adipose tissue, an elevation in
MCP-1 is associated with an increased ability to attract macrophages, which reinforces
the inflammatory cycle [25]. In endothelial cells, leptin secreted from adipose tissue enhances MCP-1 synthesis
[47]. At this point, it is not clear why the SUB group had a greater reduction in MCP-1
compared to the EGG group. However, the reduction of MCP-1 observed in both groups,
although not significant in the EGG group, may be the result of decreased circulating
leptin due to weight loss in the current study. We have also observed that subjects
in both groups reduced levels of leptin following the CRD intervention (Ratliff and
Fernandez, unpublished observations).

VCAM-1 and ICAM-1

VCAM-1 and ICAM-1 levels did not change throughout the intervention for either group.
Circulating adhesion molecules play an active role in the atherogenic process by adhering
circulating leukocytes into vessel walls. Proteolysis of membrane bound molecules
releases soluble forms of VCAM-1 and ICAM-1 into the bloodstream, which can serve
as markers of endothelial inflammation [48,49]. Endothelial VCAM-1 and ICAM-1 are upregulated in response to TNF-α [50]. In the current study, there was no change in TNF-α observed in either group, which
may result in the unchanged serum VCAM-1 and ICAM-1 values. A previous study found
that caloric restriction in conjunction with exercise for one year lowered plasma
VCAM-1 and ICAM-1 in overweight women [51]. The brief duration of the current study, gender, or the lack of structured exercise
might also explain why there was not a reduction in cellular adhesion molecules for
either group.

Conclusion

We conclude that daily intake of 3 eggs while following a CRD results in a significant
decrease in CRP and a more pronounced increase in adiponectin, thus, improving the
inflammatory profile. These findings suggest a role of eggs due to their cholesterol
and antioxidant content in achieving maximum benefits with carbohydrate restricted
diets on certain inflammatory cytokines.

Competing interests

Supported by a grant from the American Egg Board-Egg Nutrition Center to MLF.

Authors' contributions

JR was responsible for data acquisition, analysis, and interpretation and for drafting
of the manuscript. GM was responsible for data acquisition, analysis and interpretation
and critical revision of the manuscript. MP was responsible for data analysis and
critical revision of the manuscript. JSV was responsible for study conception and
design, data collection, analysis and interpretation and for critical revision of
the manuscript. MLF was responsible for study conception and design, data collection,
analysis and interpretation, and for critical revision of the manuscript. All authors
read and approved the final manuscript.

Acknowledgements

The authors wish to thank the Egg Nutrition Center for funding this study.